Methods for high-throughput and quantitative proteome analysis

Abstract

The invention provides methods for identifying and quantifying polypeptides in a sample. The methods include the steps of labeling peptides in a polypeptide sample with an isotope tag; adding a plurality of peptide standards to the polypeptide sample, wherein the peptide standards are labeled with an isotopically distinct version of the isotope tag; resolving the labeled sample and standard peptides into a plurality of fractions; analyzing the resolved fractions using mass spectrometry; identifying an isotope-tagged sample peptide in an analyzed fraction; and determining the amount of the identified isotope-tagged sample peptide in the analyzed fraction by comparison to the amount of isotope tagged standard peptide in the same fraction.

1. A method of identifying and quantifying polypeptides in a sample, comprising the steps of:
 (a) cleaving a polypeptide sample to generate sample peptides; 
 (b) labeling the sample peptides with a first isotope tag to provide a sample of a first isotopically-labeled peptide; 
 (c) chemically synthesizing a plurality of peptide standards of known sequences, wherein the chemically synthesized peptide standards of known sequences comprise sequences of peptides generated by the same cleaving as in step (a); 
 (d) labeling the chemically synthesized peptide standards with a second isotope tag, wherein the second isotope tag labels the chemically synthesized peptide standards with an isotopically distinct version of the first isotope tag, thereby generating a plurality of isotopically-labeled chemically synthesized peptide standards; 
 (e) adding the plurality of the isotopically-labeled chemically synthesized peptide standards of known sequences to the isotopically-labeled sample peptides, thereby generating a mixture of isotopically-labeled sample peptides and isotopically-labeled chemically synthesized peptide standards; 
 (f) resolving the mixture of labeled sample peptides and labeled chemically synthesized peptide standards into a plurality of resolved fractions; 
 (g) analyzing the resolved fractions by mass spectrometry; 
 (h) identifying an isotopically-labeled sample peptide in an analyzed fraction from step (g); and 
 (i) determining the amount of the identified isotopically-labeled sample peptide in the analyzed fraction by comparing the amount of the identified isotopically-labeled sample peptide to the amount of the isotopically-labeled chemically synthesized peptide standard in the analyzed fraction. 
 
     
     
       2. The method of  claim 1 , wherein the analyzing by mass spectrometry comprises depositing the plurality of fractions onto a mass spectrometry sample plate. 
     
     
       3. The method of  claim 1 , wherein a known absolute amount of each of the known synthetic peptide standards is added to the peptide sample. 
     
     
       4. The method of  claim 1 , wherein the cleaving of the polypeptide sample comprises cleaving with a protease. 
     
     
       5. The method of  claim 4 , wherein the protease is trypsin. 
     
     
       6. The method of  claim 1 , wherein the polypeptide sample is obtained from a body fluid selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, urine, saliva, seminal plasma, and pancreatic juice. 
     
     
       7. A method of identifying and quantifying polypeptides in a sample, comprising the steps of:
 (a) cleaving a polypeptide sample to generate sample peptides; 
 (b) labeling the sample peptides with a first isotope tag to provide a sample of a first isotopically-labeled peptide; 
 (c) synthesizing a plurality of peptide standards of known sequences by recombinantly expressing the peptide standards, wherein the peptide standards of known sequences comprise sequences of peptides generated by the same cleaving as in step (a); 
 (d) labeling the peptide standards with a second isotope tag, wherein the second isotope tag labels the peptide standards with an isotopically distinct version of the first isotope tag, thereby generating a plurality of isotopically-labeled recombinantly expressed peptide standards; 
 (e) adding a plurality of the isotopically-labeled peptide standards of known sequences to the isotopically-labeled sample peptides, thereby generating a mixture of isotopically-labeled sample peptides and isotopically-labeled peptide standards; 
 (f) resolving the mixture of labeled sample peptides and labeled peptide standards into a plurality of resolved fractions; 
 (g) analyzing the resolved fractions by mass spectrometry; 
 (h) identifying an isotopically-labeled sample peptide in an analyzed fraction from step (g); and 
 (i) determining the amount of the identified isotopically-labeled sample peptide in the analyzed fraction by comparing the amount of the identified isotopically-labeled sample peptide to the amount of the isotopically-labeled peptide standard in the analyzed fraction. 
 
     
     
       8. The method of  claim 7 , wherein each of the synthetic peptide standards is recombinantly expressed as a single peptide product. 
     
     
       9. The method of  claim 7 , wherein each of the synthetic peptide standards is recombinantly expressed as a concatenated peptide product. 
     
     
       10. The method of  claim 7 , wherein the analyzing by mass spectrometry comprises depositing the plurality of fractions onto a mass spectrometry sample plate. 
     
     
       11. The method of  claim 7 , wherein a known absolute amount of each of the known synthetic peptide standards is added to the peptide sample. 
     
     
       12. The method of  claim 7 , wherein the cleaving of the polypeptide sample comprises cleaving with a protease. 
     
     
       13. The method of  claim 12 , wherein the protease is trypsin. 
     
     
       14. The method of  claim 7 , wherein the polypeptide sample is obtained from a body fluid selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, urine, saliva, seminal plasma, and pancreatic juice. 
     
     
       15. A method of identifying and quantifying polypeptides in a sample, comprising the steps of:
 (a) cleaving a polypeptide sample to generate sample peptides; 
 (b) labeling the sample peptides with a first isotope tag to provide a sample of a first isotopically-labeled peptide; 
 (c) chemically synthesizing a plurality of peptide standards of known sequences, wherein the chemically synthesized peptide standards of known sequences comprise sequences of peptides generated by the same cleaving as in step (a); 
 (d) labeling the chemically synthesized peptide standards with a second isotope tag, wherein the second isotope tag labels the chemically synthesized peptide standards with an isotopically distinct version of the first isotope tag, thereby generating a plurality of isotopically-labeled chemically synthesized peptide standards; 
 (e) adding a known absolute amount of a plurality of the isotopically-labeled chemically synthesized peptide standards of known sequences to the isotopically-labeled sample peptides, thereby generating a mixture of isotopically-labeled sample peptides and isotopically-labeled chemically synthesized peptide standards; 
 (f) resolving the mixture of labeled sample peptides and labeled chemically synthesized peptide standards into a plurality of resolved fractions; 
 (g) analyzing the resolved fractions by mass spectrometry; 
 (h) identifying an isotopically-labeled sample peptide in an analyzed fraction from step (g); and 
 (i) determining the amount of the identified isotopically-labeled sample peptide in the analyzed fraction by comparing the amount of the identified isotopically-labeled sample peptide to the amount of the isotopically-labeled chemically synthesized peptide standard in the analyzed fraction. 
 
     
     
       16. The method of  claim 15 , wherein the analyzing by mass spectrometry comprises depositing the plurality of fractions onto a mass spectrometry sample plate. 
     
     
       17. The method of  claim 15 , wherein the cleaving of the polypeptide sample comprises cleaving with a protease. 
     
     
       18. The method of  claim 17 , wherein the protease is trypsin. 
     
     
       19. The method of  claim 15 , wherein the polypeptide sample is obtained from a body fluid selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, urine, saliva, seminal plasma, and pancreatic juice. 
     
     
       20. A method of identifying and quantifying polypeptides in a sample, comprising the steps of:
 (a) cleaving a polypeptide sample to generate sample peptides; 
 (b) labeling the sample peptides with a first isotope tag, to provide a sample of a first isotopically-labeled peptide; 
 (c) synthesizing a plurality of peptide standards of known sequences by recombinantly expressing the peptide standards, wherein the peptide standards of known sequences comprise sequences of peptides generated by the same cleaving as in step (a); 
 (d) labeling the peptide standards with a second isotope tag, wherein the second isotope tag labels the peptide standards with an isotopically distinct version of the first isotope tag, thereby generating a plurality of isotopically-labeled peptide standards; 
 (e) adding a known absolute amount of a plurality of the isotopically-labeled peptide standards of known sequences to the isotopically-labeled sample peptides, thereby generating a mixture of isotopically-labeled sample peptides and isotopically-labeled peptide standards; 
 (f) resolving the mixture of labeled sample peptides and labeled peptide standards into a plurality of resolved fractions; 
 (g) analyzing the resolved fractions by mass spectrometry; 
 (h) identifying an isotopically-labeled sample peptide in an analyzed fraction from step (g); and 
 (i) determining the amount of the identified isotopically-labeled sample peptide in the analyzed fraction by comparing the amount of the identified isotopically-labeled sample peptide to the amount of the isotopically-labeled peptide standards in the analyzed fraction. 
 
     
     
       21. The method of  claim 20 , wherein each of the synthetic peptide standards is recombinantly expressed as a single peptide product. 
     
     
       22. The method of  claim 20 , wherein each of the synthetic peptide standards is recombinantly expressed as a concatenated peptide product. 
     
     
       23. The method of  claim 20 , wherein the analyzing by mass spectrometry comprises depositing the plurality of fractions onto a mass spectrometry sample plate. 
     
     
       24. The method of  claim 20 , wherein the cleaving of the polypeptide sample comprises cleaving with a protease. 
     
     
       25. The method of  claim 24 , wherein the protease is trypsin. 
     
     
       26. The method of  claim 20 , wherein the polypeptide sample is obtained from a body fluid selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, urine, saliva, seminal plasma, and pancreatic juice. 
     
     
       27. A method for identifying and quantifying polypeptides in a sample, comprising the steps of:
 (a) labeling a polypeptide sample with a first isotope tag to provide a sample of a first isotopically-labeled polypeptide; 
 (b) cleaving the first isotopically-labeled polypeptide sample to generate isotopically-labeled sample peptides; 
 (c) chemically synthesizing a plurality of peptide standards of known sequences, wherein the chemically synthesized peptide standards of known sequences comprise sequences of peptides generated by the same cleaving as in step (b); 
 (d) labeling the chemically synthesized peptide standards with a second isotope tag, wherein the second isotope tag labels the chemically synthesized peptide standards with an isotopically distinct version of the first isotope tag, thereby generating a plurality of isotopically-labeled chemically synthesized peptide standards; 
 (e) adding the plurality of the isotopically-labeled chemically synthesized peptide standards of known sequences to the isotopically-labeled sample peptides, thereby generating a mixture of isotopically-labeled sample peptides and isotopically-labeled chemically synthesized peptide standards; 
 (f) resolving the mixture of labeled sample peptides and labeled chemically synthesized peptide standards into a plurality of resolved fractions; 
 (g) analyzing the resolved fractions by mass spectrometry; 
 (h) identifying an isotopically-labeled sample peptide in an analyzed fraction from step (g); and 
 (i) determining the amount of the identified isotopically-labeled sample peptide in the analyzed fraction by comparing the amount of the identified isotopically-labeled sample peptide to the amount of the isotopically-labeled chemically synthesized peptide standard in the analyzed fraction. 
 
     
     
       28. A method of identifying and quantifying polypeptides in a sample, comprising the steps of:
 (a) labeling a polypeptide sample with a first isotope tag to provide a sample of a first isotopically-labeled polypeptide; 
 (b) cleaving the first isotopically-labeled polypeptide sample to generate isotopically-labeled sample peptides; 
 (c) synthesizing a plurality of peptide standards of known sequences by recombinantly expressing the peptide standards, wherein the peptide standards of known sequences comprise sequences of peptides generated by the same cleaving as in step (b); 
 (d) labeling the peptide standards with a second isotope tag, wherein the second isotope tag labels the peptide standards with an isotopically distinct version of the first isotope tag, thereby generating a plurality of isotopically-labeled recombinantly expressed peptide standards; 
 (e) adding a plurality of the isotopically-labeled peptide standards of known sequences to the isotopically-labeled sample peptides, thereby generating a mixture of isotopically-labeled sample peptides and isotopically-labeled peptide standards; 
 (f) resolving the mixture of labeled sample peptides and labeled peptide standards into a plurality of resolved fractions; 
 (g) analyzing the resolved fractions by mass spectrometry; 
 (h) identifying an isotopically-labeled sample peptide in an analyzed fraction from step (g); and 
 (i) determining the amount of the identified isotopically-labeled sample peptide in the analyzed fraction by comparing the amount of the identified isotopically-labeled sample peptide to the amount of the identified isotopically-labeled peptide standard in the analyzed fraction. 
 
     
     
       29. A method of identifying and quantifying polypeptides in a sample, comprising the steps of:
 (a) labeling a polypeptide sample with a first isotope tag to provide a sample of a first isotopically-labeled polypeptide; 
 (b) cleaving the first isotopically-labeled polypeptide sample to generate isotopically-labeled sample peptides; 
 (c) chemically synthesizing a plurality of peptide standards of known sequences, wherein the chemically synthesized peptide standards of known sequences comprise sequences of peptides generated by the same cleaving as in step (b); 
 (d) labeling the chemically synthesized peptide standards with a second isotope tag, wherein the second isotope tag labels the chemically synthesized peptide standards with an isotopically distinct version of the first isotope tag, thereby generating a plurality of isotopically-labeled chemically synthesized peptide standards; 
 (e) adding a known absolute amount of a plurality of the isotopically-labeled chemically synthesized peptide standards of known sequences to the isotopically-labeled sample peptides, thereby generating a mixture of isotopically-labeled sample peptides and isotopically-labeled chemically synthesized peptide standards; 
 (f) resolving the mixture of labeled sample peptides and labeled chemically synthesized peptide standards into a plurality of resolved fractions; 
 (g) analyzing the resolved fractions by mass spectrometry; 
 (h) identifying an isotopically-labeled sample peptide in an analyzed fraction from step (g); and 
 (i) determining the amount of the identified isotopically-labeled sample peptide in the analyzed fraction by comparing the amount of the identified isotopically-labeled sample peptide to the amount of the isotopically-labeled chemically synthesized peptide standard in the analyzed fraction. 
 
     
     
       30. A method of identifying and quantifying polypeptides in a sample, comprising the steps of:
 (a) labeling a polypeptide sample with a first isotope tag to provide a sample of a first isotopically-labeled polypeptide; 
 (b) cleaving the first isotopically-labeled polypeptide sample to generate isotopically-labeled sample peptides; 
 (c) synthesizing a plurality of peptide standards of known sequences by recombinantly expressing the peptide standards, wherein the peptide standards of known sequences comprise sequences of peptides generated by the same cleaving as in step (b); 
 (d) labeling the peptide standards with a second isotope tag, wherein the second isotope tag labels the peptide standards with an isotopically distinct version of the first isotope tag, thereby generating a plurality of isotopically-labeled peptide standards; 
 (e) adding a known absolute amount of a plurality of the isotopically-labeled peptide standards of known sequences to the isotopically-labeled sample peptides, thereby generating a mixture of isotopically-labeled sample peptides and isotopically-labeled peptide standards; 
 (f) resolving the mixture of labeled sample peptides and labeled peptide standards into a plurality of fractions; 
 (g) analyzing the resolved fractions by mass spectrometry; 
 (h) identifying an isotopically-labeled sample peptide in an analyzed fraction from step (g); and 
 (i) determining the amount of the identified isotopically-labeled sample peptide in the analyzed fraction by comparison to the amount of isotopically-labeled peptide standards in the analyzed fraction.